<i>In Silico</i> Prediction and Mining of Exporters for Secretory Bioproduction of Terpenoids in <i>Saccharomyces cerevisiae</i>

Liu, Jiaheng; Wang, Xinglong; Jin, Ke; Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Liu, Long

doi:10.1021/acssynbio.2c00673

Cited by 4 publications

(5 citation statements)

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“…The original strain, S. cerevisiae S6, was chosen because it had previously been modified to overproduce squalene by overexpressing the key rate-limiting enzymes that limit the MVA pathway’s rate of production, such as tHMG1 (truncated hydroxymethylglutaryl-CoA reductase), IDI (isopentenyl diphosphate delta-isomerase), and ERG20 (farnesyl pyrophosphate synthetase). First, OSC (oxidosqualene cyclase), AtLUP1 from Arabidopsis thaliana, was integrated into the genome of S6 to produce lupeol (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae

Tang,

Xu,

Liu

et al. 2024

ACS Synth. Biol.

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Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

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Section: Resultsmentioning

confidence: 99%

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae

Tang,

Xu,

Liu

et al. 2024

ACS Synth. Biol.

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“…As the transport is equilibrative, the compounds will not accumulate inside the cell. In case of toxicity, the yeast possesses a variety of ATP-driven ABC transporters for efficient multidrug efflux (Egner and Kuchler, 1996;Ortiz et al, 1997;Prasad and Goffeau, 2012;Liu et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

“…The prevalence of promiscuous equilibrative transporters has significant implications for strain development in biotechnology. Transporter engineering has shown notable improvements in yeast cell factories for various compounds such as resveratrol (Pan et al, 2008;Vos et al, 2015), squalene (Liu et al, 2023), muconic acid (Pereira et al, 2019;Wang et al, 2021), and betaxanthin (Wang et al, 2021). Several reviews provide additional insights into this progress [see Kell et al (2015), Jezierska and Van Bogaert (2017), Kell (2018), van der Hoek and Borodina Belew et al (2022)].…”

Section: Discussionmentioning

confidence: 99%

“…Resveratrol and muconic acid have low solubility in water and readily precipitate during the process. Squalene is a waterinsoluble terpenoid that requires a biphasic fermentation to maintain high titers (Liu et al, 2023). Notably though, if transport can be changed to happen with higher energetic efficiency, then significant improvements in the cell factories by transporter engineering can be achieved.…”

Section: Discussionmentioning

confidence: 99%

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Deorphanizing solute carriers in Saccharomyces cerevisiae for secondary uptake of xenobiotic compounds

Møller-Hansen,

Sáez-Sáez,

van der Hoek

et al. 2024

Front. Microbiol.

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The exchange of small molecules between the cell and the environment happens through transporter proteins. Besides nutrients and native metabolic products, xenobiotic molecules are also transported, however it is not well understood which transporters are involved. In this study, by combining exo-metabolome screening in yeast with transporter characterization in Xenopus oocytes, we mapped the activity of 30 yeast transporters toward six small non-toxic substrates. Firstly, using LC–MS, we determined 385 compounds from a chemical library that were imported and exported by S. cerevisiae. Of the 385 compounds transported by yeast, we selected six compounds (viz. sn-glycero-3-phosphocholine, 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, acrylic acid, 2-benzoxazolol) for characterization against 30 S. cerevisiae xenobiotic transport proteins expressed in Xenopus oocytes. The compounds were selected to represent a diverse set of chemicals with a broad interest in applied microbiology. Twenty transporters showed activity toward one or more of the compounds. The tested transporter proteins were mostly promiscuous in equilibrative transport (i.e., facilitated diffusion). The compounds 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, and sn-glycero-3-phosphocholine were transported equilibratively by transporters that could transport up to three of the compounds. In contrast, the compounds acrylic acid and 2-benzoxazolol, were strictly transported by dedicated transporters. The prevalence of promiscuous equilibrative transporters of non-native substrates has significant implications for strain development in biotechnology and offers an explanation as to why transporter engineering has been a challenge in metabolic engineering. The method described here can be generally applied to study the transport of other small non-toxic molecules. The yeast transporter library is available at AddGene (ID 79999).

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“…Lycopene’s high hydrophobicity means it accumulates within the cell membrane, which affects cellular function upon high-level production. Several studies have been emerging to resolve this issue via membrane engineering − and two-phase extractive fermentation. − Though in situ extraction of lycopene will help achieve a very competitive biotechnological process for lycopene production, the inability of the organic phase or the extractant to fully extract lycopene from the cellular membrane is the potential bottleneck. Thus, engineering the cell membrane for easy penetration of the extractant will resolve this menace.…”

Section: Introductionmentioning

confidence: 99%

Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli

Fordjour,

Bai,

et al. 2023

ACS Synth. Biol.

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Lycopene biosynthesis is frequently hampered by downstream processing hugely due to its inability to be secreted out from the producing chassis. Engineering cell factories can resolve this issue by secreting this hydrophobic compound. A highly permeable E. coli strain was developed for a better release rate of lycopene. Specifically, the heterologous mevalonate pathway and crtEBI genes from Corynebacterium glutamicum were overexpressed in Escherichia coli BL21 (DE3) for lycopene synthesis. To ensure in situ lycopene production, murein lipoprotein, lipoprotein NlpI, inner membrane permease protein, and membrane-anchored protein in TolA-TolQ-TolR were deleted for improved membrane permeability. The final strain, LYC-8, produced 438.44 ± 8.11 and 136.94 ± 1.94 mg/L of extracellular and intracellular lycopene in fed-batch fermentation. Both proteomics and lipidomics analyses of secreted outer membrane vesicles were perfect indicators of hypervesiculation. Changes in the ratio of saturated fatty acids, unsaturated fatty acids, and cyclopropane fatty acids coupled with the branching and acyl chain lengths altered the membrane fatty acid composition. This ensured membrane fluidity and permeability for in situ lycopene release. The combinatorial deletion of these genes altered the cellular morphology. The structural and morphological changes in cell shape, size, and length were associated with changes in the mechanical strength of the cell envelope. The enhanced lycopene production and secretion mediated by improved membrane permeability established a cell lysis-free system for an efficient releasing rate and downstream processing, demonstrating the importance of vesicle-associated membrane permeability in efficient lycopene production.

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In Silico Prediction and Mining of Exporters for Secretory Bioproduction of Terpenoids in Saccharomyces cerevisiae

Cited by 4 publications

References 59 publications

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae

Deorphanizing solute carriers in Saccharomyces cerevisiae for secondary uptake of xenobiotic compounds

Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli

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